The overall goal of this project is to understand how telomere proteins, DNA damage response factors and replication factors cooperate to replicate and protect telomeres. Telomeres exist as protective DNA-protein complexes that are essential to maintain genome stability. Failure of the complex to reform after telomere replication leads to chromosome fusions, aneuploidy and cell death;defects that lead to human disease. Replication of a telomere involves a number of steps beyond simple duplication of the double-stranded telomeric DNA. These include processing of the DNA to generate an overhang on the 3'G-rich strand and G-strand extension by telomerase. During replication, the telomere is transiently recognized as DNA damage and it recruits proteins such as ATM, ATR, MRN and the Rad9/Rad1/Hus1 checkpoint clamp. These damage response factors seem to play an important, but poorly understood role in telomere replication. Likewise, while the steps involved in telomere replication are well established, the mechanistic details are far from clear. The proposed research addresses how the Pot1/Tpp1 heterodimer, the replication protein A (RPA) and the Rad9/Rad/1/Hus1 clamp interface with each other and with telomerase to ensure that a functional telomere is reassembled after DNA replication. The role of ATR signaling in this process is also explored. During the previous grant period we made a conditional chicken DT40 cell line (Er-Pot1) and found that a key function of the telomere protein Pot1 is to prevent the telomere from activating a DNA damage checkpoint during late S/G2 of the cell cycle. We also showed that Pot1 is required to restrict telomere growth. We will now build on these findings by using a mix of in vivo and in vitro approaches to: (1) Dissect the contribution of Pot1/Tpp1 dimerization, shelterin binding and Tpp1/telomerase interactions to telomere protection and length regulation. (2) Explore RPA function at vertebrate telomeres. (3) Determine the role of the Rad9/Rad1/Hus1 clamp and ATR in telomere maintenance. The common theme is how the presence of these three single-strand binding proteins at the G-overhang coordinates and regulates telomere replication. Telomeres are the protective DNA-protein caps at chromosomes ends that prevent chromosome fusions and degradation of the terminal DNA sequence. When a chromosome is duplicated, it takes a series of specialized steps beyond simple DNA replication to ensure that a functional, fully protected telomere is regenerated. The proposed research seeks to understand how specialized telomere proteins cooperate with DNA repair factors and replication proteins to ensure that these steps occur correctly.